Korean J Physiol Pharmacol.  2018 Jan;22(1):43-51. 10.4196/kjpp.2018.22.1.43.

Epigenetic modification of α-N-acetylgalactosaminidase enhances cisplatin resistance in ovarian cancer

• Ha YN
• Sung HY
• Yang SD
• Chae YJ
• Ju W
• Ahn JH

• KMID: 2398554
• DOI: http://doi.org/10.4196/kjpp.2018.22.1.43

Abstract

Although cisplatin is one of the most effective antitumor drugs for ovarian cancer, the emergence of chemoresistance to cisplatin in over 80% of initially responsive patients is a major barrier to successful therapy. The precise mechanisms underlying the development of cisplatin resistance are not fully understood, but alteration of DNA methylation associated with aberrant gene silencing may play a role. To identify epigenetically regulated genes directly associated with ovarian cancer cisplatin resistance, we compared the expression and methylation profiles of cisplatin-sensitive and -resistant human ovarian cancer cell lines. We identified α-Nacetylgalactosaminidase (NAGA) as one of the key candidate genes for cisplatin drug response. Interestingly, in cisplatin-resistant cell lines, NAGA was significantly downregulated and hypermethylated at a promoter CpG site at position +251 relative to the transcriptional start site. Low NAGA expression in cisplatin-resistant cell lines was restored by treatment with a DNA demethylation agent, indicating transcriptional silencing by hyper-DNA methylation. Furthermore, overexpression of NAGA in cisplatin-resistant lines induced cytotoxicity in response to cisplatin, whereas depletion of NAGA expression increased cisplatin chemoresistance, suggesting an essential role of NAGA in sensitizing ovarian cells to cisplatin. These findings indicate that NAGA acts as a cisplatin sensitizer and its gene silencing by hypermethylation confers resistance to cisplatin in ovarian cancer. Therefore, we suggest NAGA may be a promising potential therapeutic target for improvement of sensitivity to cisplatin in ovarian cancer.

Keyword

Cisplatin resistance; DNA methylation; Ovarian cancer; α-N-acetylgalactosaminidase

MeSH Terms

Antineoplastic Agents
Cell Line
Cisplatin*
DNA
DNA Methylation
Epigenomics*
Gene Silencing
Humans
Methylation
Ovarian Neoplasms*
Antineoplastic Agents
Cisplatin
DNA

Figure

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  Fig. 1 NAGA expression is down-regulated in cisplatin-resistant cell lines.NAGA mRNA expression was determined by gene expression microarray (A) and RT-qPCR (B) in eight ovarian cancer cell lines. Error bars represent standard deviation (SD) of triplicate measurements. Statistical analyses were performed using a t-test.

• 

  Fig. 2 A CpG site is hypermethylated within the NAGA promoter in cisplatin-resistant cell lines.The DNA methylation status of CpG sites within the NAGA promoter region was quantified using the Illumina HumanMethylation 450 BeadChip in eight ovarian cancer cell lines. The Illumina HumanMethylation 450 BeadChip includes eleven CpG sites within NAGA promoter region, which is located at −656, −524, −498, −471, −299, −271, −260, −255, −157, +12 and +251 from TSS (A). The DNA methylation status of the CpG site located +251 relative to the TSS is illustrated in (B). The statistical differences between cisplatin-sensitive and -resistant cell lines were determined by a t-test. TSS, transcription start site.

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  Fig. 3 NAGA mRNA expression is restored by demethylation in cisplatin-resistant cell lines.Eight ovarian cancer cell lines were treated with 5-aza-2′-deoxycytidine and NAGA mRNA expression was measured by RT-qPCR. The relative expression levels to dimethyl sulfoxide (DMSO)-treated controls in cisplatin-sensitive and cisplatin-resistant cell lines (A), or in eight individual cisplatin-resistant cell lines (B) are shown. Data are presented as the mean±SD from three independent experiments. Statistical analyses were performed using a t-test. Con, control; Aza, 5-aza-2′-deoxycytidine.

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  Fig. 4 Overexpression of NAGA sensitizes ovarian cancer cells to cisplatin.Cisplatin-resistant SK-OV-3 cells and cisplatin-sensitive TOV-112D cells were transiently transfected with EGFP and NAGA expression constructs, and overexpression of NAGA was confirmed by RT-qPCR in SK-OV-3 cells (A) and TOV-112D cells (B). Cytotoxicity in response to cisplatin in NAGA-overexpressing SK-OV-3 (C) and TOV-112D cells (D) was assayed after 48 h cisplatin treatment using an MTT assay. Data are represented as the mean±SD from three independent experiments. The statistical difference was determined by a t-test.

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  Fig. 5 Transient depletion of NAGA by siRNA causes ovarian cancer cell resistance to cisplatin.Cisplatin-resistant SK-OV-3 cells and cisplatin-sensitive TOV-112D cells were transiently transfected with siNC and siNAGA. After 24 h of transfection, knockdown of NAGA was confirmed by RT-qPCR in SK-OV-3 cells (A) and TOV-112D cells (B). Cytotoxicity in response to cisplatin in NAGA-depleted SK-OV-3 cells (C) and TOV-112D cells (D) was assayed after 48 h treatment with cisplatin using an MTT assay. All data is shown as the mean±SD of triplicate measurements. The statistical difference was determined by a t-test. siNC, non-targeting control siRNA; siNAGA, NAGA siRNA.

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  Fig. 6 Regulation of cisplatin-induced apoptosis by ectopic expression of NAGA.Cisplatin-resistant SK-OV-3 cells were transiently transfected with EGFP and NAGA expression constructs or with siNC and siNAGA. After 24 h of transfection, culture medium was replaced with fresh medium containing 30 µM cisplatin. Following 10 h of cisplatin treatment, cells were collected for western blot analysis to determine the Caspace-3 activation. All data is shown as the mean±SD of triplicate measurements. The statistical difference was determined by a t-test. siNC, non-targeting control siRNA; siNAGA, NAGA siRNA.

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